WO2009008556A1 - Nanofilm de protéine, flexible et autonome, procédé de fabrication et application - Google Patents

Nanofilm de protéine, flexible et autonome, procédé de fabrication et application Download PDF

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Publication number
WO2009008556A1
WO2009008556A1 PCT/JP2008/062979 JP2008062979W WO2009008556A1 WO 2009008556 A1 WO2009008556 A1 WO 2009008556A1 JP 2008062979 W JP2008062979 W JP 2008062979W WO 2009008556 A1 WO2009008556 A1 WO 2009008556A1
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protein
film
membrane
thin
self
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PCT/JP2008/062979
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English (en)
Japanese (ja)
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Xinshen Peng
Izumi Ichinose
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National Institute For Materials Science
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Priority to US12/452,570 priority Critical patent/US8741152B2/en
Priority to EP08778271.0A priority patent/EP2168669B1/fr
Priority to CN2008800238968A priority patent/CN101918119B/zh
Publication of WO2009008556A1 publication Critical patent/WO2009008556A1/fr
Priority to US12/654,931 priority patent/US8828239B2/en
Priority to US14/290,327 priority patent/US20140263036A1/en

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D71/00—Semi-permeable membranes for separation processes or apparatus characterised by the material; Manufacturing processes specially adapted therefor
    • B01D71/06—Organic material
    • B01D71/74—Natural macromolecular material or derivatives thereof
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D67/00—Processes specially adapted for manufacturing semi-permeable membranes for separation processes or apparatus
    • B01D67/0002—Organic membrane manufacture
    • B01D67/0006—Organic membrane manufacture by chemical reactions
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D69/00—Semi-permeable membranes for separation processes or apparatus characterised by their form, structure or properties; Manufacturing processes specially adapted therefor
    • B01D69/12—Composite membranes; Ultra-thin membranes
    • B01D69/122—Separate manufacturing of ultra-thin membranes
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D69/00—Semi-permeable membranes for separation processes or apparatus characterised by their form, structure or properties; Manufacturing processes specially adapted therefor
    • B01D69/14—Dynamic membranes
    • B01D69/141—Heterogeneous membranes, e.g. containing dispersed material; Mixed matrix membranes
    • B01D69/142—Heterogeneous membranes, e.g. containing dispersed material; Mixed matrix membranes with "carriers"
    • B01D69/144—Heterogeneous membranes, e.g. containing dispersed material; Mixed matrix membranes with "carriers" containing embedded or bound biomolecules
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D2323/00—Details relating to membrane preparation
    • B01D2323/30—Cross-linking
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01D—SEPARATION
    • B01D2325/00—Details relating to properties of membranes
    • B01D2325/04—Characteristic thickness
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S977/00—Nanotechnology
    • Y10S977/70—Nanostructure
    • Y10S977/701—Integrated with dissimilar structures on a common substrate
    • Y10S977/702—Integrated with dissimilar structures on a common substrate having biological material component
    • Y10S977/705—Protein or peptide
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S977/00—Nanotechnology
    • Y10S977/84—Manufacture, treatment, or detection of nanostructure
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S977/00—Nanotechnology
    • Y10S977/84—Manufacture, treatment, or detection of nanostructure
    • Y10S977/89—Deposition of materials, e.g. coating, cvd, or ald

Definitions

  • the present invention is capable of rapidly and simply separating (or concentrating) molecules having a relatively small molecular weight (about 100 million) as well as a large molecular weight. “Soft and self-supporting ultrathin (nano) or thin
  • the present invention relates to a protein membrane J.
  • the present invention also relates to a method for producing the protein membrane or an application of the protein membrane.
  • membranes are used for the production of drinking water from seawater, the purification of industrial wastewater, the recovery of precious components, the concentration, purification or fractionation of polymer mixtures in the food and pharmaceutical industry, and the separation of gases and vapors. Therefore, it is widely applied in practice.
  • Membranes are also an important component in energy conversion systems, artificial organs or drug delivery devices.
  • the widespread use of membranes in separation operations is to prepare a favorable combination of membranes that are highly selective, resulting in high purity of the separation material, low cost of handling, and high permeability (ie, membranes). It is difficult to reduce not only the high flux but also the membrane area and manufacturing cost.
  • the high flow rate (flux) of the membrane is a key basic performance that determines the cost of the membrane system.
  • ultra-thin (several tens of nm) self-supporting films have been reported (Yang, H. et al, 1996; Mamedov, AA et al, 2002; others), and are used for sensor actuators. ing. However, these separation operations are not reported because of functional or workability defects.
  • the exception to this is the first example of using ultra-thin nanomethylene for size-based separation of macromolecules, prepared by an accurate silicon deposition and etching technique and a high temperature (approximately 700 ° C) thermal annealing process. There is only a report by Striemer and his collaborators using a 15 nm thick free-standing silicon film (Striemer. CC et al, 2007).
  • Non-Patent Document 1 a simple filtration and exfoliation technique to create negatively charged dye molecules (see Non-Patent Document 1), DNA (see Non-Patent Document 2), or positively charged metal hydroxide nanostrands
  • Non-Patent Document 2 a simple filtration and exfoliation technique to create negatively charged dye molecules
  • DNA a DNA
  • Non-Patent Document 2 a DNA
  • metal hydroxide nanostrands We have developed a general synthesis method for ultra-thin, self-supporting mesoporous membranes with a nano-composite structure, which has a fibrous nanocomposite structure. Unfortunately, however, these fibrous nanocomposite films were brittle and easily broken due to the poor chemical stability of metal hydroxide nanostrands.
  • Non-Patent Document 1 Lo, Y.-H., Huang, J., Ichinose, I. "Bundle -like assemblies of cadmium hydroxide nanostrands and anionic dyes” J. Am. Chem. Soc. 127, 8296-8297 ( 2005).
  • Non-Patent Document 2 Ichinose, I., Huang, J., Lou, ⁇ . ⁇ . "Electrostatic trapping of double-strand DNA by using cadmium hydroxide nanostrands” Nano Lett. 5, 97-100 (2005).
  • Non-Patent Document 3 Ichinose, I., Krashima, K., Kunitake. T. "Spontaneous formation of cadmium hydroxide nanostrands in water” J. Am. Chem. Soc. 126, 7162-7163 (2004).
  • Non-patent document 4 Lo, Y.-H. et al. "Formation of positively charged copper hydroxide nanostrands and their structural characterization” Chem. Mater. 18, 1795-1782 (2006) Disclosure of the invention
  • the present inventors further studied a method of coating positively charged metal hydroxide nanostrands with proteins. Fortunately, the protein in the fibrous composite is covalently cross-linked with dartal aldehyde (GA), and then the inorganic nanostrands are removed, notably, a strong, flexible and self-supporting ultrathin (nano ) We have succeeded in developing a pure or thin protein membrane. Means for solving the problem
  • the present invention provides a soft and self-supporting ultra-thin (nano or nm scale) or thin protein film, wherein the protein is bound (cross-linked) with a bifunctional cross-linking agent.
  • “membrane” has the same meaning as “film”
  • “self-supporting” has the same meaning as “self-supporting”.
  • the present invention also provides a method for producing the soft and self-supporting ultra-thin (nano) or thin protein film. The manufacturing method includes the following steps:
  • Step of forming metal hydroxide nanostrand That is, dilute a metal (Cd, Cu or Zn) nitrate or hydrochloride to neutral or weakly basic pH Hold and spontaneously form the metal (Cd, Cu or Zn) hydroxide nanostrands;
  • Step of obtaining a composite nano-liper made of protein and the above metal hydroxide nanostrand That is, the obtained metal (C d, Cu or Zn) is mixed with the protein solution.
  • Step of cross-linking That is, the protein contained in the composite nanofiber is bonded (cross-linked) with a bifunctional cross-linking agent;
  • Step of removing metal hydroxide That is, metal (Cd, 011 or 211) hydroxide nanostrand is removed from the reaction product.
  • the present invention also provides some applications of the self-supporting ultra-thin or thin protein membranes.
  • one layer is the above protein film, and the other layer is a thin molecular film formed by depositing a predetermined molecule on the protein film and cross-linking with a bifunctional cross-linking agent.
  • a self-supporting thin film consisting of two layers is provided by the present invention. The invention's effect
  • the soft and self-supporting ultra-thin (nano) or thin protein membrane of the present invention is novel.
  • the resulting protein membrane has a uniform thickness of 25 nm, a diameter of 7.5 cm, and a diameter / thickness ratio of 3, 0 0 0, 0
  • the film showed 0 0 (such a high ratio has never been reported before).
  • the soft and self-supporting ultra-thin (nano) or thin protein membranes of the present invention can be applied to size-selective separation of small molecular weight molecules (about 1,00 or less). They also have very high capacities (molar ratios) for efficient separation of molecules under pH control and for reversible adsorption and desorption of dye molecules with varying pH. Can be applied.
  • the soft and self-supporting ultra-thin (nano) or thin protein film can be easily produced.
  • One layer of the present invention is the above-described protein film, and the other layer is a thin molecular film formed by depositing a predetermined molecule on the protein film and cross-linking with a bifunctional cross-linking agent.
  • the two-layered self-supporting thin film is a novel multilayer film and can be used differently from the soft self-supporting ultra-thin (nano) or thin protein film.
  • Figure 1 is a schematic diagram showing a typical manufacturing process for ultra-thin (nano) or thin, self-supporting protein membranes.
  • Figure 2 is a TEM planar image of a nanofibrous film of ferritin Z hydroxylated domum nanostrand after the Tachibana reaction.
  • FIG. 3 is a TEM planar image of the ferritin film after removal of hydroxylated domum nanostrands from the nanofibrous film of ferritin / hydroxylated dimethyl nanostrand shown in FIG.
  • Figure 4 is a copy of a photograph of a self-supporting ferritin membrane with a diameter of 7.5 cm.
  • Fig. 5 shows (a) SEM cross-sectional image of ferritin / hydroxylated dominum nanostrand film (thickness: 40 nm) before removal of hydroxylated dominum nanostrand.
  • B SEM plane It is a statue.
  • Figure 6 shows (A) a SEM cross-sectional image of a ferritin film (thickness: 40 nm) after removal of cadmium hydroxide nanostrands, and (b) a SEM plane image.
  • Figure 7 shows E D X spectra recorded from the crosslinked film before (Before) and after (After) removal of hydroxylated domum nanostrands.
  • Figure 8 shows the FTIR spectrum of the membrane after (i) before cross-linking (no-cross link) and (ii) after removal of hydroxylating power nano-strands after cross-linking (cross-link remove CdOH). .
  • Figure 9 shows the loading-unloading of ferritin and apoferritin membranes with and without hydroxylated domum nanostrands. It is a typical example of a curve.
  • Figure 10 shows a typical UV-visible spectrum and photocopy showing the concentration performance for PC / Cu.
  • Figure 11 shows a typical UV-visible spectrum and photocopy showing Absorption of Evans Blue when using an apoferritin membrane (thickness: 300 nm, diameter: 3.2 cm). It is.
  • Figure 12 shows a typical UV-visible spectrum and photographic image showing Evanspur's desorption when using an apoferritin membrane (thickness: 300 nm, diameter: 3.2 cm). It is a copy.
  • Figure 13 is a typical example of a photoluminescence (PL) spectrum showing fluorescent dye molecules desorbed by a ferritin film.
  • the photograph (copy) of the insert was taken under light irradiation with a wavelength of 375 nm.
  • the structure of the fluorescent dye molecule (OPTAT) is shown in the lower right corner of the figure.
  • Fig. 14 is a typical SEM image of a PAM AM film (thickness: 4.6 ⁇ m) formed on an apoferritin film.
  • (B) shows an enlarged image of the part surrounded by the dotted line in (a).
  • the production method of the present invention includes the following steps.
  • step (4) crosslinking step
  • a step of removing the composite nanobuyper made of protein and metal hydroxide it is possible to add a step of removing the composite nanobuyper made of protein and metal hydroxide.
  • various kinds of proteins can be used. Examples are shown below using ferritin, apoferritin, cytochrome c, myoglobin, and gnorecosoxidase (and, of course, other proteins can be used). Not only a single protein but also a mixed protein can be used, but it is preferable to use a single protein because it can be expected to be uniform.
  • a typical manufacturing process scheme for a self-supporting ultra-thin (nano) or thin protein membrane is shown in Figure 1.
  • a composite nanofiber dispersion made of protein and metal hydroxide nanostrand is generated.
  • the resulting dispersion is filtered on a filter such as a polycarbonate (PC) membrane with a pore size of 200 nm (porosity is about 10%) to form a composite nanofibrous film.
  • PC polycarbonate
  • the obtained film is treated with a solution of a bifunctional crosslinking agent (for example, 10% by weight aqueous solution of glutaraldehyde) for a sufficient time to complete the crosslinking reaction.
  • a bifunctional crosslinking agent for example, 10% by weight aqueous solution of glutaraldehyde
  • Figure 1 shows an example of peeling these cross-linked nanofibrous composite films.
  • the film-equipped filter can be immersed in alcohol (for example, ethanol) to obtain a self-supporting crosslinked film.
  • the crosslinked film having self-supporting properties is immersed in a mineral acid aqueous solution such as a hydrochloric acid solution to remove the metal hydroxide nanostrand.
  • a mineral acid aqueous solution such as a hydrochloric acid solution
  • the excess metal ions and hydrochloric acid are washed away with purified water.
  • a pure (ie, free of metal hydroxide), free-standing protein membrane floating in water can be obtained.
  • These membranes can be stored in alcohol for further application or evaluation.
  • the thickness of the protein membrane can be easily controlled in the range of 10 nm to 10 ⁇ m by adjusting the volume of the fibrous composite liquid to be filtered (see Example 3, Table 1).
  • the thickness of the protein membrane is preferably 15 nm to 100 nm, more preferably 20 nm to 100 nm. nm.
  • the thickness of the protein membrane and the time for the filtering operation linearly depend on the volume of the filtrate.
  • the size (diameter) of the protein membrane is not limited. This is because the diameter of the protein membrane is basically determined by the size (inner diameter) of the funnel.
  • the above self-supporting ultra-thin (nano) or thin protein membrane has various applications. As mentioned above, one application is that one to three layers are the protein membrane, and the other one is to deposit a given molecule on the protein membrane and crosslink with a bifunctional crosslinker. This is the preparation of a self-supporting thin film consisting of two layers of thin molecular films.
  • the “predetermined molecule” various molecules having a known structure such as a synthetic polymer can be used which have a molecular weight large enough to prevent passage through the protein membrane passage.
  • a synthetic polymer for example, a dendrimer having a terminal amino group can be used.
  • a polyamidoamine having a molecular weight of about 2,00 or more (sized so as not to pass through the protein membrane passage) is preferably used.
  • Ponolephine p Tonorensenorephonic acid, 8-octanoloxypyrene mono-1,3,6-trisulfonic acid trisodium salt, copper lid Cyanine tetrasulfonic acid
  • glucose oxidase, cytochrome c, myoglobin, horse spleen ferritin (76 mg / ml solution) and apoferritin (38 mg / ml solution) were purchased from Sigma-Aldrich. Deionized water (18.2 ⁇ ) was produced by Millipore's Direct Q system and used throughout the experiment.
  • PC polycarbonate
  • Nutarepore Nutarepore, Watman
  • Alumina membrane Alignment, pore size 0.2 / 2 111, diameter 2.5 cm, thickness 60 m
  • the equipment and method used are as follows. The obtained film is a scanning electron microscope.
  • a hydroxylated dimethyl nanostrand having a positive charge like a polymer was prepared by the method described in the literature (see Non-Patent Documents 2 to 4). Briefly, dilute caustic soda solution or aminoethanol solution (2 mM, 20 mL) is poured into 4 mM nitric acid aqueous solution 2 OmL, mixed quickly, stirred for several minutes, Munanostrands were prepared.
  • Protein (ferritin, apoferritin, cytochrome c, myoglobin, or glucose oxidase) was added to the obtained hydroxylated dimethyl nanostrand dispersion and stirred for 30 minutes.
  • the mixture consists of 1 mL of a 3.8 mg / mL protein solution and 20 mL of a cadmium hydroxide dispersion.
  • the mixture is 6.4 mg Zm It is made by mixing lm L of L protein solution and 20 mL of hydroxylated dimethyl nanostrand dispersion.
  • a predetermined amount of the mixture was filtered under a gauge pressure (differential pressure) of 90 KPa on a polycarbonate membrane (diameter of membrane Z funnel used for filtration: 3.2 cm).
  • the membrane was immersed in a 10% by weight dartal aldehyde aqueous solution and allowed to undergo a crosslinking reaction at room temperature for 1 hour.
  • the nanofibrous composite film after the crosslinking reaction was obtained by immersing the PC membrane with film in ethanol and peeling it off.
  • the obtained self-supporting membrane was immersed in a 1 O mM hydrochloric acid solution for 3 hours to remove inorganic nanostrands, and then excess force of dimethyl ions and hydrochloric acid was washed away using Milli-Q purified water. .
  • five kinds of pure self-supporting proteins (ferritin, apoferritin, cytochrome c, myoglobin, or glucose oxidase) each floating in water were obtained.
  • Fig. 2 shows a TEM image of a nanofibrous film of ferritin Z hydroxylating power domum nanostrand after the cross-linking reaction. From this image, the fibrous structure is clear, and most of the protein is in nanostrand. You can see that they are gathering along. In this image, the ferritin protein appears as a black spot with a diameter of about 8 nm due to the iron compound core of ferritin. Hydroxylation power Dominum nanostrand looks like a fiber structure with a thickness of about 2 nm.
  • Figure 3 is a TEM image of a pure ferritin film after removal of hydroxylated domum nanostrand from the nanofibrous film of ferritin / hydroxylated domumnostrand shown in Figure 2. In this image, the fibrous structure has disappeared, which means that the nanostrand has been completely removed.
  • Figure 4 is a copy of a photograph of a self-supporting ferritin membrane with a diameter of 7.5 cm. The diameter of this membrane is equal to the size of the funnel used for filtration.
  • Figure 5 shows SEM cross-sectional images (a) and SEM planar images (b) of the ferritin Z hydroxide cadmium nanostrand film (thickness: 40 nm) before removal of hydroxylated domumanostrand. These are the SEM cross-sectional image (a) and SEM plane image (b) of the ferritin film (thickness: 40 nm) after the removal of hydroxylated dimethyl nanostrand.
  • the characteristic peaks are almost the same, but the peak intensity is stronger in the ferritin film than in the natural pheritin.
  • the same peak position is due to the fact that no new functional groups were introduced into the membrane during the cross-linking reaction.
  • hydroxylation strength domumanostrand does not adversely affect the protein membrane by removing it, it has the same properties as hydration strength domumanostrand, and if it does, it is safer to hydroxylate.
  • Copper nanostrands can also be used as nanostrands such as zinc oxide nanostrands.
  • Sex protein membranes can be prepared (data not shown here)
  • the self-sustainability is the same as in Example 1 except that the filtration time and Z or the amount of filtrate are changed, and the diameter of the membrane Z funnel used is 1.7 cm, 3.2 cm, or 7.5 cm.
  • An ultra-thin pure protein membrane was prepared. The results are shown in Table 1.
  • V m i xture a is a ferritin or apoferritin protein 3.8 mg / mi (6.4 mg Zm 1 for other proteins) solution 2 m 1 and cadmium hydroxide nanostrand solution 40 m 1 This means the filtration capacity of the mixture at that time.
  • the thickness of the protein film was measured from a cross-sectional SEM image.
  • the obtained film was evaluated by SEM image and TEM image (not shown here) together with macroscopic observation (or photograph). From these results, it can be seen that the thickness of the protein membrane and the time of the filtration operation linearly depend on the amount of filtrate.
  • films were obtained with three diameters of 1.7 cm, 3.2 cm, and 7.5 cm. And in the case of ferritin, the diameter was 1.7 cm, the thinnest 40 nm, and the diameters were 3.2 cm and 7.5 cm, each 60 nm. The thickest one had a diameter of 1.7 cm and a filtration time of 1 hour, reaching 400 nm. That is, in the case of ferritin, the thickness of the ferritin film can be controlled in the range from 40 nm to 400 nm.
  • the resulting apoferritin ultrathin protein membrane was a uniform thickness of 25 ⁇ m even when the diameter was 7.5 cm (No. 1 2). . The ratio of diameter to thickness reached 3, 0 0 0, 0 0 0 times.
  • the TEM image shows that the film is very soft on the nm scale.
  • This extreme mac-scale flexibility was supported by inhaling an apoferritin free-standing membrane with a diameter of 7.5 c Hi into a pipette with a 0.8 mm diameter tip. It was surprising that this protein membrane was able to reversibly pass through a hole as small as 8770 times its area (size). This is due to the flexibility and extreme thinness of the protein membrane.
  • b nl indicates the number of measurement points, and nl, n2 and n3 mean measurements at three points.
  • c Avg means the average of the measured values at 3 points.
  • the values of hardness and Young's modulus of the ferritin film and apoferritin film are the same as the previously reported natural protein film of glyoxal cross-linked structure such as gelatin, soybean, casein or sojumucaseinate (Vaz, CM et al, J. Mater. Sci. : Mater, in Medicine 14, 789-796 (2003), 10 times higher than the value of soy protein membrane with a dartal aldehyde crosslinked structure (Chabba, S. et al, J. Mater. Sci. 40, 6263- 6273, 2005).
  • Example 3 Application of the protein membrane of the present invention to separation, concentration, absorption or desorption The separation characteristics of these self-supporting pure protein membranes can be transmitted through molecules with various sizes, charge states and pH characteristics. We studied by studying. The filtration operation was performed under a pressure of 90 kPa. The capacity of the molecule (including the solution) is 20 m 1. The flow rate is equal to the permeate volume normalized by the effective area and operating time. The transmission characteristics were monitored by UV visible absorption spectra recorded for the solution before and after filtration, as well as the top solution. The results are summarized in Table 3.
  • FIGS. 12a and 12b show the experimental results of release. After 2.5 h (1 50 min), 83.5% of the Evans blue molecules were released from the membrane into the solution. As time increased, more dye molecules were released into the solution.
  • the maximum rate of the first release circle was 94.5%, which was similar to the release rate of DY molecules (data not shown). However, after the first capture and release circle, the release efficiency was 99.4% for the later circle.
  • the capture and release operation is due to the induction of the charge properties of the apoferritin membrane by pH. (Ie, when the pH of the solution is lower than the isoelectric point of apoferritin 4.4, the membrane is positively charged; conversely, when the pH of the solution is higher than 4.4, The protein membrane will be negatively charged. Therefore, in solutions with pH lower than 4.4, negatively charged molecules will be trapped in the membrane by electrostatic forces. Otherwise (the pH of the solution is higher than 4.4), negatively charged dye molecules will be driven off the membrane by repulsion.
  • Example 4 The preparation of the protein film of the present invention for the preparation of a self-supporting thin film composed of two layers, that is, one layer of a protein film and the other layer of a molecular thin film formed thereon. Another application
  • the method for preparing the protein (apoferritin) membrane is the same as that described in Example 1.
  • a 1.9 ⁇ m-thick apoferritin thin film placed on a polycarbonate membrane having a pore size of 200 nxn was used in a suction filtration system.
  • Dendrimer P AMAM molecule generally 4, molecular weight: 1 4 2 1 5, diameter: 4.5 nm, number of amino groups on the surface: 64, purchased from Sigma-Aldrich
  • 2% methanol solution 2 ml was filtered through the suction filtration system under a gauge pressure (differential pressure) of 90 kPa.
  • FIG. 14 shows a typical SEM image of a 4.6 ⁇ m thick PAMAM film formed on the surface of a 1.9 ⁇ m thick apoferritin film.
  • the thickness of the PAMAM membrane can be controlled by the amount of filtrate of the PAMAM solution.
  • other molecular membranes can also be prepared by using the same filtration operation and cross-linking process based on protein membranes. This method provides a simple way to construct functional molecular thin films for various applications such as separation.

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Abstract

L'invention vise à proposer un “film ultramince (nano) ou mince de protéine pure, qui est très résistant, flexible et autonome” avec lequel des molécules relativement petites (masse moléculaire : environ 1000 Da) peuvent être rapidement et facilement séparées (ou concentrées). Le film ultramince (nano) ou mince de protéine pure, qui est très résistant, flexible et autonome peut être obtenu par un procédé comportant les étapes suivantes : (1) tout en maintenant une solution diluée d'un nitrate ou chlorhydrate d'un métal (Cd, Cu ou Zn) à une valeur de pH neutre ou faiblement basique, amener la formation spontanée de nano-brins d'hydroxyde du métal (Cd, Cu ou Zn); (2) mélanger les nano-brins d'hydroxyde du métal (Cd, Cu ou Zn) ainsi obtenus avec une solution de protéine pour donner des nanofibres composites composées de la protéine avec les nanos-brins d'hydroxyde du métal (Cd, Cu ou Zn); (3) filtrer la dispersion des nanofibres composites ainsi obtenues à travers un filtre; (4) réticuler la protéine contenue dans les nanofibres composites avec un agent réticulant bifonctionnel; et (5) retirer les nanofibres d'hydroxyde du métal (Cd, Cu ou Zn) à partir du mélange réactionnel tel que décrit ci-dessus.
PCT/JP2008/062979 2007-07-11 2008-07-11 Nanofilm de protéine, flexible et autonome, procédé de fabrication et application WO2009008556A1 (fr)

Priority Applications (5)

Application Number Priority Date Filing Date Title
US12/452,570 US8741152B2 (en) 2007-07-11 2008-07-11 Flexible free-standing ultrathin or thin protein membrane, its fabrication method and application
EP08778271.0A EP2168669B1 (fr) 2007-07-11 2008-07-11 Nanofilm de protéine, flexible et autonome et procédé de fabrication
CN2008800238968A CN101918119B (zh) 2007-07-11 2008-07-11 柔软的自立性的蛋白质纳米薄膜、其制造方法和应用
US12/654,931 US8828239B2 (en) 2007-07-11 2010-01-08 Flexible free-standing ultrathin or thin protein membrane, its fabrication method and application
US14/290,327 US20140263036A1 (en) 2007-07-11 2014-05-29 Flexible free-standing ultrathin or thin protein membrane

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JP2007181832 2007-07-11
JP2007-181832 2007-07-11
JP2007290238A JP5278939B2 (ja) 2007-07-11 2007-11-08 柔らかで自立性があるタンパク質ナノ薄膜、その製造法及び応用
JP2007-290238 2007-11-08

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EP2604330A1 (fr) * 2011-12-16 2013-06-19 Samsung Electronics Co., Ltd Film semi-perméable et membrane de séparation comprenant un matériau nanoporeux et son procédé de fabrication
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US8741152B2 (en) 2014-06-03
CN101862609A (zh) 2010-10-20
CN101918119A (zh) 2010-12-15
EP2168669B1 (fr) 2016-11-23
US8828239B2 (en) 2014-09-09
EP2179779A2 (fr) 2010-04-28
JP5278939B2 (ja) 2013-09-04
US20100140163A1 (en) 2010-06-10
JP2009131725A (ja) 2009-06-18
EP2168669A1 (fr) 2010-03-31
EP2179779B1 (fr) 2016-11-23
CN101918119B (zh) 2013-07-03
EP2179779A3 (fr) 2011-11-02
CN101862609B (zh) 2013-07-03
US20100219120A1 (en) 2010-09-02
US20140263036A1 (en) 2014-09-18
EP2168669A4 (fr) 2011-11-02

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